Techniques such as molecular cloning, Southern blotting, Northern blotting and in situ hybridization exploit the specificity and sensitivity of nucleic acid hybridization. These procedures routinely employ polynucleotide probes of high specific radioactivity coupled with autoradiographic detection methods.
Fluorescence in situ hybridization (FISH) is an important and powerful diagnostic tool that helps bridge the resolution gap between chromosome analysis and molecular techniques. The ability to label DNA probe sets and to hybridize them to cytogenetic preparations demonstrates that this technology can be used to detect minute chromosomal abnormalities. The high resolution of this technique allows direct visualization of single copy sequences. Because each chromosome occupies a distinct domain or discrete focal territory in the interphase nucleus (Lichter, P., et al., PNAS USA. 85:9664-9668, 1988; Manuelidis, L. Hum. Genet. 71:288-293, 1985; Cremer, T. et al., Exp. Cell Res. 176:199-220, 1988), a discrete FISH signal is obtained in most nuclei for each specific chromosome present. Therefore, an interphase nucleus with three copies of chromosome 13 will show three chromosome 13 FISH signals while a normal disomic nucleus will have 2 signals. Appropriate probe sets based on cosmid contigs that are chromosome specific can be used to enumerate chromosomes in prenatal diagnostics. Trisomic karyotypes have been diagnosed by FISH procedures (Lichter, P., et al., PNAS USA. 85:9664-9668, 1988; Klinger, K., et al., Am. J. Hum. Genet. 51:55-65, 1992). It has been shown that variations in sample preparation for FISH can have major effects on hybridizability and signal quality (Jordan, C. A. 1990. In situ hybridization in cells and tissue sections: a study of myelin gene expression during CNS myelination and remyelination. In: Cheselet M-F (ed) In situ hybridization histochemistry. CRC Press, Boca Raton, pp 39-70; Lichter, J. P., Jaunch, A., Cremer, T., and Ward, D.C. 1990. Detection of Down syndrome by in situ hybridization with chromosome 21 specific DNA probes. In: Patterson, D. (ed) Molecular genetics of chromosome 21 and Down syndrome. Wiley-Liss, New York, pp69-78.; McNeil, et al., Genet. Anal. Tech. Appl. 8:41-58, 1991). Optimal FISH parameters are also dependent upon the cell type.
FISH is frequently used in conjunction with chromosome analysis. Most chromosome spreads analyzed in clinical laboratories are derived from abundant sources of artificially induced or naturally dividing cell types (e.g., lymphocytes, amniocytes, bone marrow). In situations where the target cell is not abundant or if it is quiescent or infrequently dividing (e.g., rare cancer cells in Minimal Residual Disease or fetal cells in maternal cell circulation), more cells are accessible to analysis using interphase FISH than analysis based on chromosomes.
Clearly, a need exists for a method of sample capture and analysis that minimizes cell loss (e.g., by reducing the number of cell concentration steps or centrifugation which may be harmful to the cell) and maximizes the access to potentially informative target material. It is apparent that different cell types require different processing steps for optimal FISH results. In addition, a need exists for a process that enables simple, quick, and simultaneous processing of multiple samples.